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. 2022 Jul;29(7):1318-1334.
doi: 10.1038/s41418-022-01025-9. Epub 2022 Jun 20.

Ca2+-mediated mitochondrial inner membrane permeabilization induces cell death independently of Bax and Bak

Giovanni Quarato  1 , Fabien Llambi  2   3 , Cliff S Guy  2 , Jaeki Min  4   5 , Marisa Actis  4 , Huan Sun  6 , Shilpa Narina  7 , Shondra M Pruett-Miller  7 , Junmin Peng  6 , Zoran Rankovic  4 , Douglas R Green  8
Affiliations

Ca2+-mediated mitochondrial inner membrane permeabilization induces cell death independently of Bax and Bak

Giovanni Quarato et al. Cell Death Differ. 2022 Jul.

Abstract

The ability of mitochondria to buffer a rapid rise in cytosolic Ca2+ is a hallmark of proper cell homeostasis. Here, we employed m-3M3FBS, a putative phospholipase C (PLC) agonist, to explore the relationships between intracellular Ca2+ imbalance, mitochondrial physiology, and cell death. m-3M3FBS induced a potent dose-dependent Ca2+ release from the endoplasmic reticulum (ER), followed by a rise in intra-mitochondrial Ca2+. When the latter exceeded the organelle buffering capacity, an abrupt mitochondrial inner membrane permeabilization (MIMP) occurred, releasing matrix contents into the cytosol. MIMP was followed by cell death that was independent of Bcl-2 family members and inhibitable by the intracellular Ca2+ chelator BAPTA-AM. Cyclosporin A (CsA), capable of blocking the mitochondrial permeability transition (MPT), completely prevented cell death induced by m-3M3FBS. However, CsA acted upstream of mitochondria by preventing Ca2+ release from ER stores. Therefore, loss of Ca2+ intracellular balance and mitochondrial Ca2+ overload followed by MIMP induced a cell death process that is distinct from Bcl-2 family-regulated mitochondrial outer membrane permeabilization (MOMP). Further, the inhibition of cell death by CsA or its analogues can be independent of effects on the MPT.

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Conflict of interest statement

The authors declare no competing interests. DRG consults for Inzen Therapeutics and Ventus Therapeutics.

Figures

Fig. 1
Fig. 1. m-3M3FBS induces intracellular calcium mobilization and mitochondrial calcium overload.
a FLIM intracellular calcium quantification in HeLa cells after treatment with 25 μM m-3M3FBS. Each dashed line represents one single cell in the field. The continuous bold line represents the mean signal from all the cells in the field. Cells were pre-labeled with 4 μM of Oregon Green™ 488 BAPTA-1, AM. Data are representative of n = 4 independent experiments. b Intracellular Ca2+ spike quantification using FLIM. Each dot represents the highest Δ[Ca2+] value recorded for each cell. Δ[Ca2+] was calculated as maximum amplitude of [Ca2+] oscillation from [Ca2+]t=0. Cells were pre-labeled with 4 μM of Oregon Green™ 488 BAPTA-1, AM. Data are from n = 4 independent experiments. c Confocal microscopy analysis showing matrix mitochondrial calcium accumulation using mito-GCaMP3 in HeLa cells after treatment with 25 μM m-3M3FBS; magenta arrows indicate cells undergoing transitory mitochondrial matrix calcium accumulation. Data are representative of n = 3 independent experiments. Scale bar = 50 μm. d Intracellular calcium mobilization in HeLa cells after treatment with 25 μM m-3M3FBS (as fluorescence F/F0 arbitrary units) with a schematic representation of the results. Upper panel: cytosolic calcium (cyto-GCaMP3) vs ER lumen calcium (ER-GECO). Middle panel: mitochondrial matrix calcium (mito-GCaMP3) vs ER lumen calcium (ER-GECO). Lower panel: matrix mitochondrial calcium (mito-GCaMP3) vs cytosolic calcium (cyto-GECO). Each dashed line represents one single cell in the field. The continuous bold line represents the mean signal from all the cells in the field. Data are representative of n = 4 independent experiments. e Matrix mitochondrial calcium accumulation using mito-GCaMP3 and mitochondrial transmembrane potential in HeLa cells after treatment with 25 μM m-3M3FBS; 200 nM TMRE was preincubated 5’ before beginning of the experiment. Data are representative of n = 3 independent experiments. f OCR and ECAR of HeLa cells treated as indicated by the arrows using a Seahorse XFe bioanalyzer. “Glu” = 10 mM glucose and “Inh.” (inhibitors) = 0.5 μM rotenone + 0.5 μM antimycin A (for OCR) and 50 mM 2-deoxy-glucose (for ECAR), as detailed in Methods section. m-3M3FBS was used at 25 μM. Error bars represent the SEM from the mean of n ≥ 13 samples. Data are representative of n = 3 independent experiments.
Fig. 2
Fig. 2. Mitochondrial calcium overload induces mitochondrial inner membrane permeabilization and cell death.
a SIM images of HeLa cells after 3 min treatment with 25 μM m-3M3FBS: cyan: TOM20 (outer mitochondrial membrane), red: TFAM-mCherry (mitochondrial matrix); green arrows indicate release of mitochondrial matrix protein into the cytosol. Representative image of n = 3 independent fields per condition. Scale bar = 10 μm. b Immunoblot analysis showing release of soluble mitochondrial proteins into the cytosol after treatment with 25 μM m-3M3FBS or a combination of Bcl-2/Bcl-xL (5 μM ABT-737) and Mcl-1 (5 μM S63845) inhibitors for 20 and 40 min followed by digitonin permeabilization in HeLa cells. To synchronize the onset of MOMP, cells were primed with a sublethal dose of UV irradiation (5 mJ/cm2) 16 h prior to ABT-737 plus S63845 addition. Data are representative of n = 3 independent experiments. c TEM analysis of HeLa cells after treatment with 25 μM m-3M3FBS for 15 min; green arrows indicate mitochondria, magenta arrows indicate ER. Scale bar = 5 μm. Scale bar for magnified images = 0.5 μm. d Kinetic analysis of cell death in HeLa cells after treatment with m-3M3FBS using an IncuCyte imaging system. Error bars represent the SD from the mean of triplicate samples. Data are representative of n = 4 independent experiments. e Representative IncuCyte images at 1 h intervals of PI-stained HeLa cells after treatment with 25 μM m-3M3FBS. Related to d. Scale bar = 200 μm.
Fig. 3
Fig. 3. MIMP is a kinetically distinct process than MOMP and it is not regulated by Bcl-2 family members.
a Confocal microscopy analysis showing release of the IMS proteins omi-mCherry (red) and the Matrix proteins mito-GCaMP3 (green) in HeLa cells after 10 min treatment with 5 μM ABT-737 or 25 μM m-3M3FBS. To synchronize the onset of MOMP, cells were primed with a sublethal dose of UV irradiation (5 mJ/cm2) 16 h prior to ABT-737 addition. Data are representative of n = 3 independent experiments. Scale bar = 40 μm. b Release kinetics of IMS proteins (omi-mCherry, red) and Matrix proteins (mito-GCaMP3, green) in HeLa cells after treatment with ABT-737 or m-3M3FBS. Experimental conditions are the same as in a. Regions were drawn around cells, and punctate/diffuse indices were plotted and displayed relative to the start of the treatment (time point 0). Each dashed line represents one single cell in the field (n ≥ 9). The continuous bold line represents the mean signal from all the cells in the field for each marker. Data are representative of n = 3 independents experiments. c Cell death quantification of HeLa WT and Bax,Bak DKO cells after 6 h of treatment with 25 μM m-3M3FBS or 5 μM ABT-737 + 5 μM S63845 using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. d Kinetic analysis of HeLa cells treated with 25 μM m-3M3FBS or 5 μM ABT-737 + 5 μM S63845 after silencing of APAF1 using an IncuCyte imaging system. Error bars represent the SD from the mean of triplicate samples. Data are representative of n = 3 independent experiments. e Intracellular Ca2+ spike quantification using FLIM in HeLa cells overexpressing HA-Bcl-2, HA-Bcl-xL and HA-Mcl-1 after treatment with 25 μM m-3M3FBS. Each dot represents the highest Δ[Ca2+] value recorded per cell. Δ[Ca2+] was calculated as maximum amplitude of [Ca2+] oscillation from [Ca2+]t=0. Cells were pre-labeled with 4 μM of Oregon Green™ 488 BAPTA-1, AM. Data are from n = 4 independent experiments. f Kinetic analysis of cell death in HeLa cells overexpressing HA-Bcl-2, HA-Bcl-xL and HA-Mcl-1 after treatment with 25 μM m-3M3FBS. Error bars represent the SD from the mean of triplicate samples. Data are representative of n = 4 independent experiments.
Fig. 4
Fig. 4. Buffering of intracellular calcium protects from m-3M3BFS induced cell death.
a Intracellular Ca2+ spike quantification using FLIM in HeLa cells after treatment with 25 μM m-3M3FBS or 0.5 μM Thapsigargin. Each dot represents the highest Δ[Ca2+] value recorded per cell. Δ[Ca2+] was calculated as maximum amplitude of [Ca2+] oscillation from [Ca2+]t=0. Cells were pre-labeled with 4 μM of Oregon Green™ 488 BAPTA-1, AM. Data are from n = 4 independent experiments. b Mitochondrial matrix calcium (mito-GCaMP3) vs cytosolic calcium (cyto-GECO) mobilization in HeLa cells after treatment with 25 μM m-3M3FBS or 0.5 μM Thapsigargin (as fluorescence F/F0 arbitrary units). Each dashed line represents one single cell in the field. The continuous bold line represents the mean signal from all the cells in the field. Data are representative of n = 4 independent experiments. c Cell death quantification of HeLa cells after 6 h of treatment with 25 μM m-3M3FBS in combination with 20 μM BAPTA-AM (BAPTA) using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. d Mitochondrial matrix calcium quantification (mito-GCaMP3) in HeLa cells WT and expressing exogenous PVALB-myc and mito-PVALB-myc after treatment with 25 μM m-3M3FBS (as fluorescence F/F0 arbitrary units). Each dashed line represents one single cell in the field. The continuous bolder line represents the mean signal from all the cells in the field. Data are representative of n = 4 independent experiments. e Cell death quantification of HeLa cells WT and expressing exogenous PVALB-myc and mito-PVALB-myc after 6 h of treatment with 25 μM m-3M3FBS using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. f Release kinetic of IMS proteins (omi-mCherry, red) and Matrix proteins (COX8A1–29-Cerulean, blue) in HeLa cells after treatment with 150 μM Arachidonate, 0.5 μM Thapsigargin, 40 μM C2-cer. and 1 mM H2O2. Regions were drawn around cells, and punctate/diffuse indices were plotted and displayed relative to the start of the treatment (time point 0). Each dashed line represents one single cell in the field. The continuous bold line represents the mean signal from all the cells in the field for each marker. Data are representative of n = 3 independents experiments. g Cell death quantification of HeLa cells after 6 h of treatment with 150 μM Arachidonate, 0.5 μM Thapsigargin, 40 μM C2-cer. and 1 mM H2O2 using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. h Release kinetic of IMS proteins (omi-mCherry, red) and Matrix proteins (COX8A1–29-Cerulean, blue) in HeLa cells after treatment with 150 μM Arachidonate in media containing 10% FBS. Regions were drawn around cells, and punctate/diffuse indices were plotted and displayed relative to the start of the treatment (time point 0). Each dashed line represents one single cell in the field. The continuous bold line represents the mean signal from all the cells in the field for each marker. Data are representative of n = 3 independents experiments. i Cell death quantification of HeLa cells after 24 h of treatment with 150 μM Arachidonate, 0.5 μM Thapsigargin, 40 μM C2-cer. and 1 mM H2O2 using media containing 10% FBS in an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments.
Fig. 5
Fig. 5. m-3M3FBS induced MIMP and cell death does not rely on the main ER-mitochondria Ca2+ routes.
a Cell death quantification of HeLa cells after 6 h of treatment with 25 μM m-3M3FBS in combination with 20 μM Dantrolene (Dantro) and 10 μM Xestospongin C (Xe. C) using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. b Intracellular Ca2+ spike quantification using FLIM in HeLa WT and sgITPR TKO pool cells after treatment with 100 μM Histamine or 0.5 μM Thapsigargin or 25 μM m-3M3FBS. Each dot represents the highest Δ[Ca2+] value recorded per cell. Δ[Ca2+] was calculated as maximum amplitude of [Ca2+] oscillation from [Ca2+]t=0. Cells were pre-labeled with 4 μM of Oregon Green™ 488 BAPTA-1, AM. Data are from n = 4 independent experiments. c Cell death quantification of HeLa WT and sgITPR TKO pool cells after 6 h of treatment with 25 μM m-3M3FBS using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. d Kinetic analysis of cell death in HeLa cells treated with 25 μM m-3M3FBS after silencing of MCU and OPA1 using an IncuCyte imaging system. Error bars represent the SD from the mean of triplicate samples. Data are representative of n = 3 independent experiments. e Cell death quantification of HeLa cells after 6 h of treatment with 25 μM m-3M3FBS and silencing of LETM1 using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. f Cell death quantification of HeLa WT, MCU−/− LETM1−/− clone 1 and MCU−/− LETM1−/− clone 2 cells after 6 h of treatment with 25 μM m-3M3FBS using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. g Mitochondrial matrix calcium quantification (mito-GCaMP3) in HeLa WT and sgMCU/sgLETM1 DKO pool after treatment with 0.5 μM Thapsigargin or 25 μM m-3M3FBS (as fluorescence F/F0 arbitrary units). Each dashed line represents one single cell in the field. The continuous bold line represents the mean signal from all the cells in the field. Data are representative of n = 4 independent experiments. h Cell death quantification of HeLa WT and Bax,Bak DKO cells after 6 h of treatment with 25 μM m-3M3FBS or 5 μM ABT-737 + 5 μM S63845 in combination (A/S) with 40 μM qVD and/or 40 μM Calpain Inhibitor III (Calp.i). Error bars represent the SD from the mean of triplicate samples. Error bars represent the SD from the mean of n = 3 independent experiments. i Cell death quantification of HeLa WT and Bax,Bak DKO cells after 6 h of treatment with 25 μM m-3M3FBS or 5 μM ABT-737 + 5 μM S63845 in combination (A/S) with 2 μM jasplakinolide (Jaspla) or 10 μM docetaxel (Doce) or 5 μM withaferin A (W.A). Error bars represent the SD from the mean of n = 3 independent experiments.
Fig. 6
Fig. 6. MIMP is inhibited by Cyclosporine A independently of Cyclophilins.
a Cell death quantification of HeLa cells after 6 h of treatment with 25 μM m-3M3FBS in combination with 20 μM Cyclosporin A (CsA) or 20 μM NIM-811 or 20 μM Alisporivir using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. b Clonogenic survival of HeLa cells after treatment with 25 μM m-3M3FBS in combination with 40 μM qVD or 20 μM CsA. Data are representative of n = 4 independent experiments. c Cell death quantification of MEFs ppif+/+ and ppif−/− after 6 h of treatment with 25 μM m-3M3FBS in combination with 20 μM CsA using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. d Clonogenic survival of MEFs ppif+/+ and ppif−/− after treatment with 25 μM m-3M3FBS in combination with 20 μM CsA. Data are representative of n = 3 independent experiments. e Cell death quantification of HeLa cells after 6 h of treatment with 25 μM m-3M3FBS and silencing of ATP5MG, using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. f Cell death quantification of HeLa cells after 6 h of treatment with 25 μM m-3M3FBS in combination with different concentrations of bongkrekic acid (BK), as indicated in the Figure, using an IncuCyte imaging system. Error bars represent the SD from the mean of n = 3 independent experiments. No statistically significant differences were observed between treated samples when compared to CTRL. g Cell death quantification of HeLa cells after 6 h of treatment with 25 μM m-3M3FBS and silencing of SPG7, VDAC1, VDAC2 and VDAC3, using an IncuCyte imaging system. Error bars represent the SD from the mean of n ≥ 2 independent experiments. No statistically significant differences were observed between treated samples when compared to NT siRNA. h Cell death in HeLa cells after 6 h of treatment with 25 μM m-3M3FBS and silencing of the peptidylprolyl isomerase (PPIase) family members, as reported in Supp. Table 4, using an IncuCyte imaging system. Error bars represent the SD from the mean of triplicate samples. Error bars represent the SD from the mean of n = 3 independent experiments. No statistically significant differences were observed between treated samples when compared to NT siRNA. i Intracellular Ca2+ spike quantification using FLIM in HeLa cells after treatment with 25 μM m-3M3FBS in combination with 20 μM CsA or 20 μM Alisporivir. Each dot represents the highest Δ[Ca2+] value recorded for each cell. Δ[Ca2+] was calculated as maximum amplitude of [Ca2+] oscillation from [Ca2+]t=0. Cells were pre-labeled with 4 μM of Oregon Green™ 488 BAPTA-1, AM. Data are from n = 4 independent experiments. j Intracellular calcium mobilization in HeLa cells after treatment with 25 μM m-3M3FBS and 20 μM CsA at single cell level, to compare to Fig. 1d (as fluorescence F/F0 arbitrary units). Upper panel: cytosolic calcium (cyto-GCaMP3) vs ER lumen calcium (ER-GECO); middle panel mitochondrial matrix calcium (mito-GCaMP3) vs ER lumen calcium (ER-GECO); lower panel matrix mitochondrial calcium (mito-GCaMP3) vs cytosolic calcium (cyto-GECO). Each dashed line represents one single cell in the field. The continuous bold line represents the mean signal from all the cells in the field. Data are representative of n = 4 independent experiments. k Kinetic analysis of cell death in HeLa cells treated with 150 μM Arachidonate in combination with 20 μM CsA. Error bars represent the SD from the mean of triplicate samples. Data are representative of n = 3 independent experiments.
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Comment in

  • Mitochondria in the line of fire.
    Marchi S, Pinton P. Marchi S, et al. Cell Death Differ. 2022 Jul;29(7):1301-1303. doi: 10.1038/s41418-022-01034-8. Epub 2022 Jul 13. Cell Death Differ. 2022. PMID: 35831625 Free PMC article. No abstract available.

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